TY - JOUR

T1 - Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

AU - Gårdsvoll, Henrik

AU - Werner, Finn

AU - Søndergaard, Leif

AU - Danø, Keld

AU - Ploug, Michael

PY - 2004

Y1 - 2004

N2 - The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1-283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS-PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.

AB - The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1-283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS-PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.

KW - Animals

KW - CHO Cells

KW - Cells, Cultured

KW - Cricetinae

KW - Cricetulus

KW - Drosophila

KW - Female

KW - Glycosylation

KW - Humans

KW - Mass Spectrometry

KW - Mutagenesis, Site-Directed

KW - Protein Binding

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Recombinant Proteins

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Surface Plasmon Resonance

KW - Urokinase-Type Plasminogen Activator

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.pep.2003.12.002

DO - 10.1016/j.pep.2003.12.002

M3 - Journal article

C2 - 15003263

VL - 34

SP - 284

EP - 295

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -